Method of measuring cell-free dna in surgical drain fluid to select adjuvant therapy

ABSTRACT

A method for detecting minimal residual disease in a subject following a cancer surgery is disclosed. The method includes obtaining a sample from the subject. The sample includes a surgical drainage. The method also includes isolating an amount of tumor-associated genetic material from the sample, sequencing the amount of tumor-associated genetic material to detect and quantify at least one tumor-associated mutation or variant in the amount of tumor-associated genetic material, and providing the at least one quantity of the at least one tumor-associated mutation or variant to a practitioner.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority from U.S. Provisional Application Ser.No. 62/948,686 filed on Dec. 16, 2019, which is incorporated herein byreference in its entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

Not applicable.

FIELD OF THE DISCLOSURE

The present disclosure generally relates to methods of diagnosing andtreating high-risk cancers of the neck and throat.

BACKGROUND OF THE DISCLOSURE

For patients undergoing surgical treatments of various cancers, thereare currently no effective methods of determine if any cancerous tissueswere left behind after surgery. As a consequence, physicians must definepost-surgery treatments such as radiation or chemo based on limitedinformation, affecting patient outcomes and quality of life.

Over the last five years, there has been growing interest in thedevelopment of liquid biopsies for cancer diagnosis and treatment.

Recently, several groups have sought to develop a cell-free DNA (cfDNA)liquid biopsy test for early detection of recurrence in HPV(+) head andneck cancer by detecting HPV DNA associated with circulating tumor cells(ctHPVDNA) in plasma. Many of these tests have relied on measurement oftumor-derived cell-free DNA (cfDNA) in plasma as a predictor of patientssusceptible to cancer or as a marker of tumor recurrence. AlthoughctHPVDNA testing in plasma is promising as an early marker ofrecurrence, it may not yet be a reliable prognostic tool but could alsobe used potentially to define the extent of treatment necessary as a wayof de-intensifying therapy.

However, the detection of plasma analytes, such as ctDNA or cfDNA, isill-suited for post-surgery monitoring for residual cancer at surgicalsites given the characteristics of the circulatory system. Given thesystemic nature of circulation, the detection of cfDNA in plasma isrelatively non-specific and does not provide the ability to determinewhether the source of the ctDNA or cfDNA is the surgical site. Further,the dilution of analytes within a patient's plasma volume results inrelatively low concentrations of analytes in plasma samples, increasingthe challenge of detection. In addition, the relatively long latency ofthe appearance of cfDNA in the plasma results in a lag between therelease of ctDNA or cfDNA from the surgical site. As a consequence,post-surgical treatments may be delayed, potentially affecting patientoutcomes and quality of life.

SUMMARY OF THE DISCLOSURE

In one aspect, a method for detecting minimal residual disease in asubject following a cancer surgery is disclosed. The method includesobtaining a sample from the subject. The sample includes a surgicaldrainage. The method also includes isolating an amount oftumor-associated genetic material from the sample, sequencing the amountof tumor-associated genetic material to detect and quantify at least onetumor-associated mutation or variant in the amount of tumor-associatedgenetic material, and providing the at least one quantity of the atleast one tumor-associated mutation or variant to a practitioner. The atleast one tumor-associated mutation or variant is indicative of minimalresidual disease in the subject. In some aspects, the amount oftumor-associated genetic material includes cell-free DNA, RNA, proteins,exosomes, and any combination thereof. In some aspects, isolating theamount of tumor-associated genetic material from the sample furtherincludes filtering the sample, centrifuging the sample, contacting thesample with a chromatography medium, and any combination thereof. Insome aspects, the method further includes selecting an additionaltreatment based on the quantity of the at least one tumor-associatedmutation or variant. In some aspects, the additional treatment isselected from radiotherapy, chemotherapy, follow-up surgery, activesurveillance with imaging, and any combination thereof. In some aspects,the tumor-associated genetic material is produced by a plurality ofcancer cells. In some aspects, the plurality of cancer cells areselected from one of oropharyngeal cancer cells, lung cancer cells,breast cancer cells, melanoma cells, colon cancer cells, thyroid cancercells, prostate cancer cells, ovarian cancer cells, testicular cancercells, penile cancer cells, cervical cancer cells, anal cancer cells,brain cancer cells, liver cancer cells, pancreatic cancer cells, andtesticular cancer cells. In some aspects, the cancer surgery is selectedfrom a resectioning surgery, a dissection surgery, an excision surgery,and any combination thereof. In some aspects, obtaining the sample fromthe subject further includes capturing a surgical drainage from adrainage tube associated with the cancer surgery. In some aspects,obtaining the sample from the subject further includes capturing asurgical drainage from the drainage tube within about 24 hours of thecancer surgery. In some aspects, the tumor-associated genetic materialincludes cell-free HPV DNA (cfDNA) associated with oropharyngeal cancercells. In some aspects, sequencing the amount of tumor-associatedgenetic material to detect and quantify at least one tumor-associatedmutation or variant further includes subjecting the sample to asequencing method selected from next generation DNA sequencing, nextgeneration RNA sequencing, next generation protein sequencing, PCR,Western blot, and any combination thereof. The method of any precedingclaim, wherein sequencing the amount of tumor-associated geneticmaterial to detect and quantify at least one tumor-associated mutationor variant in the amount of tumor-associated genetic material furthercomprises detecting and quantifying at least one HPV strain associatedwith HPV(+) oropharyngeal cancer comprising HPV16 DNA, HPV18 DNA, HPV31DNA, HPV33 fragment, HPV35 fragment, HPV45 DNA, HPV52 DNA, HPV58 DNA,and any combination thereof.

In another aspect, a method for selecting a post-operative treatment fora cancer patient in need is disclosed. The method includes obtaining asample from the subject. The sample includes a surgical drainage. Themethod also includes isolating an amount of tumor-associated geneticmaterial from the sample, sequencing the amount of tumor-associatedgenetic material to detect and quantify at least one tumor-associatedmutation or variant in the amount of tumor-associated genetic material,providing the at least one quantity of the at least one tumor-associatedmutation or variant to a practitioner, and selecting an additionaltreatment based on the quantity of the at least one tumor-associatedmutation or variant. The at least one tumor-associated mutation orvariant is indicative of minimal residual disease in the subject. Insome aspects, the amount of tumor-associated genetic material includescell-free DNA, RNA, proteins, exosomes, and any combination thereof. Insome aspects, isolating the amount of tumor-associated genetic materialfrom the sample further includes filtering the sample, centrifuging thesample, contacting the sample with a chromatography medium, and anycombination thereof. In some aspects, the additional treatment isselected from radiotherapy, chemotherapy, follow-up surgery, activesurveillance with imaging, and any combination thereof. In some aspects,the tumor-associated genetic material is produced by a plurality ofcancer cells. In some aspects, the plurality of cancer cells areselected from one of oropharyngeal cancer cells, lung cancer cells,breast cancer cells, melanoma cells, colon cancer cells, thyroid cancercells, prostate cancer cells, ovarian cancer cells, testicular cancercells, penile cancer cells, cervical cancer cells, anal cancer cells,brain cancer cells, liver cancer cells, pancreatic cancer cells, andtesticular cancer cells. In some aspects, the cancer surgery is selectedfrom a resectioning surgery, a dissection surgery, an excision surgery,and any combination thereof. In some aspects, obtaining the sample fromthe subject further includes capturing a surgical drainage from adrainage tube associated with the cancer surgery. In some aspects,obtaining the sample from the subject further includes capturing asurgical drainage from the drainage tube within about 24 hours of thecancer surgery. In some aspects, the tumor-associated genetic materialincludes cell-free HPV DNA (cfDNA) associated with oropharyngeal cancercells. In some aspects, sequencing the amount of tumor-associatedgenetic material to detect and quantify at least one tumor-associatedmutation or variant further includes subjecting the sample to asequencing method selected from next generation DNA sequencing, nextgeneration RNA sequencing, next generation protein sequencing, PCR,Western blot, and any combination thereof. The method of any precedingclaim, wherein sequencing the amount of tumor-associated geneticmaterial to detect and quantify at least one tumor-associated mutationor variant in the amount of tumor-associated genetic material furthercomprises detecting and quantifying at least one HPV strain associatedwith HPV(+) oropharyngeal cancer comprising HPV16 DNA, HPV18 DNA, HPV31DNA, HPV33 fragment, HPV35 fragment, HPV45 DNA, HPV52 DNA, HPV58 DNA,and any combination thereof.

Other objects and features will be in part apparent and in part pointedout hereinafter.

DESCRIPTION OF THE DRAWINGS

Those of skill in the art will understand that the drawings, describedbelow, are for illustrative purposes only. The drawings are not intendedto limit the scope of the present teachings in any way.

FIG. 1 is a flow chart illustrating a method of detecting minimalresidual disease after a surgery in accordance with one aspect of thedisclosure.

FIG. 2 is a schematic illustration of a method of isolating exosomesfrom a surgery drainage sample in accordance with one aspect of thedisclosure.

FIG. 3A is an image of exosomes isolated from a surgery drain fluidusing ulracentrifugation.

FIG. 3B is an image of exosomes isolated from a surgery drain fluidusing ulracentrifugation using the method illustrated in FIG. 2 .

FIG. 4A is a graph comparing levels of HPV DNA associated withcirculating tumor cells (ctHPVDNA) detected in the plasma of healthypatients, non-HP-associated cancer patients, and HPV(−)/HPV(+)oropharyngeal cancer patients.

FIG. 4B is a graph comparing levels of HPV16 DNA associated withcirculating tumor cells (ctHPV16DNA) detected in the plasma oforopharyngeal cancer patients at different tumor stages.

FIG. 4C is a graph comparing levels of HPV16 DNA associated withcirculating tumor cells (ctHPV16DNA) detected in the plasma oforopharyngeal cancer patients with different tumor stages and/or tobaccopack year (TPY) scores.

FIG. 5A is an image of an exosome prep obtained from surgery drain fluidof a patient with HPV positive oropharyngeal cancer.

FIG. 5B is an image of an exosome prep obtained from surgery drain fluidof a patient with HPV positive oropharyngeal cancer.

FIG. 5C is an image of an exosome prep obtained from surgery drain fluidof a patient with HPV positive oropharyngeal cancer.

FIG. 5D is an image of an exosome prep obtained from surgery drain fluidof a patient with HPV positive oropharyngeal cancer.

FIG. 5E is an image of an exosome prep obtained from surgery drain fluidof a patient with HPV positive oropharyngeal cancer.

FIG. 5F is an image of an exosome prep obtained from surgery drain fluidof a patient with HPV positive oropharyngeal cancer.

FIG. 5G is an image of an exosome prep obtained from surgery drain fluidof a patient with HPV positive oropharyngeal cancer.

FIG. 5H is an image of an exosome prep obtained from surgery drain fluidof a patient with HPV positive oropharyngeal cancer.

FIG. 5I is an image of an exosome prep obtained from surgery drain fluidof a patient with HPV positive oropharyngeal cancer.

FIG. 5J is an image of an exosome prep obtained from surgery drain fluidof a patient with HPV positive oropharyngeal cancer.

FIG. 6A is a graph comparing the proportion of favorable and unfavorablectHPV16DNA plasma clearance profiles in oropharyngeal cancer patientprofiles identified as low-risk and high-risk.

FIG. 6B is a graph summarizing disease progression in oropharyngealcancer patients from low/high risk populations with favorable andunfavorable ctHPV16DNA clearance profiles.

FIG. 6C is a survival curve comparing regional disease-free survivalover 24 months for oropharyngeal cancer patients from low/high riskpopulations with favorable and unfavorable ctHPV16DNA clearanceprofiles.

FIG. 7A is a graph comparing survival curves for oropharyngeal cancerpatient populations characterized as negative or positive for extranodalextensions (ENE).

FIG. 7B is a graph comparing survival curves for oropharyngeal cancerpatient populations characterized as nodal stages N1 and N2, as well asnegative or positive for extranodal extensions (ENE).

FIG. 8A is a graph comparing levels of cell-free HPV DNA obtained fromdrain fluid following neck dissection surgery for oropharyngeal cancerpatient populations characterized as nodal stages N0-N1, and nodalstages N2; differences were observed with a trend toward higher HPV DNAlevels with more aggressive disease; no statistical tests were performeddue to small sample size.

FIG. 8B is a graph comparing levels of cell-free HPV DNA obtained fromdrain fluid following neck dissection surgery for oropharyngeal cancerpatient populations characterized by extranodal extension (ENE) status;differences were observed with a trend toward higher HPV DNA levels withmore aggressive disease; no statistical tests were performed due tosmall sample size.

FIG. 9 is a graph comparing expression levels of cell-free HPV DNAobtained from drain fluid following neck dissection surgery fororopharyngeal cancer patient populations as a function of the number ofHPV+ lymph nodes; HPV DNA level in surgical drain fluid was stronglycorrelated with the number of positive lymph nodes; Spearman's rho wassignificant for a strong positive correlation between pathologicallyconfirmed positive lymph nodes and amount of HPV DNA in drain fluidfollowing neck dissection.

FIG. 10 is a graph comparing expression levels of cell-free HPV DNAobtained from ipsilateral and contralateral drain fluid followingbilateral neck dissection surgery; lower levels of HPV DNA were detectedin the contralateral neck drain with respect to the primary tumor inpatients undergoing bilateral neck dissection; number of positive lymphnodes reported on final pathology are displayed above the measuredvalues for the ipsilateral (red circle) and contralateral (blue square)drain fluid samples; HPV DNA was not detectable in any of the samplesfrom necks without nodal disease.

FIGS. 11A is an image of an exosome prep obtained from surgery drainfluid of a patient with HPV negative oropharyngeal cancer.

FIG. 11B is an image of an exosome preps obtained from surgery drainfluid of a patient with HPV negative oropharyngeal cancer.

FIG. 11C is an image of an exosome preps obtained from surgery drainfluid of a patient with HPV negative oropharyngeal cancer.

FIG. 11D is an image of an exosome preps obtained from surgery drainfluid of a patient with HPV negative oropharyngeal cancer.

FIG. 11E is an image of an exosome preps obtained from surgery drainfluid of a patient with HPV negative oropharyngeal cancer.

FIG. 11F is an image of an exosome preps obtained from surgery drainfluid of a patient with HPV negative oropharyngeal cancer.

FIG. 11G is an image of an exosome preps obtained from surgery drainfluid of a patient with HPV negative oropharyngeal cancer.

FIG. 11H is an image of an exosome preps obtained from surgery drainfluid of a patient with HPV negative oropharyngeal cancer.

FIG. 11I is an image of an exosome preps obtained from surgery drainfluid of a patient with HPV negative oropharyngeal cancer.

FIG. 11J is an image of an exosome preps obtained from surgery drainfluid of a patient with HPV negative oropharyngeal cancer.

There are shown in the drawings arrangements, which are presentlydiscussed, it being understood, however, that the present embodimentsare not limited to the precise arrangements and are instrumentalitiesshown. While multiple embodiments are disclosed, still other embodimentsof the present disclosure will become apparent to those skilled in theart from the following detailed description, which shows and describesillustrative aspects of the disclosure. As will be realized, theinvention is capable of modifications in various aspects, all withoutdeparting from the spirit and scope of the present disclosure.Accordingly, the drawings and detailed description are to be regarded asillustrative in nature and not restrictive.

DETAILED DESCRIPTION OF THE INVENTION

As the incidence of HPV(+) head and neck cancer continues to rise, thereis a need to provide objective measures of tumor burden in order tosafely provide de-intensified therapy. Currently, assessments ofextranodal extensions (ENE) and numbers of positive lymph nodes (nodestaging) represent the gold standard of prognostic measures obtainedafter surgical neck dissection, as illustrated in FIG. 7A and FIG. 7B.Both assessments require histological analysis of invasively excisedtissues.

When compared to HPV-negative head and neck cancers, HPV-positivecancers are associated with a much better prognosis (see FIGS. 6A, 6B,and 6C). Without being limited to any particular theory, patients withlow HPV16DNA levels are thought to have a higher likelihood of HPVintegration, which is associated with poor outcome in HPV(+) cancers.HPV(+) cancers are treated either with transoral surgery or withdefinitive radiation therapy, and several national trials are focused onde-intensification of treatment in order to reduce side effects.However, to date no objective measures to define inclusion in treatmentde-intensification are currently available.

Cell-free DNA and circulating tumor cells (CTCs) obtained from plasmasamples have emerged as powerful analytes for identifying resistance totherapy across a variety of cancer types. Preliminary results indicatethat plasma-detected HPV DNA may serve as a promising biomarker ofrecurrence of oropharyngeal cancer, but the reliability of this methodremains to be confirmed.

In various aspects, drain fluid samples collected after a surgical neckdissection treatment serve as a more proximal indicator of thepost-surgical condition of oropharyngeal tissues, in particular withrespect to HPV expression, relative to plasma samples. In various otheraspects, HPV DNA in drain fluid could serve as an adjunct objectivemeasure to traditional histopathology and plasma-based cfDNA.

In various aspects, HPV DNA is measured within samples from drain fluidcollected following neck dissection surgery. In other aspects, thepresence or absence of residual HPV DNA after surgery are be used as amore proximal liquid biomarker than corresponding plasma-detected HPVDNA, and could help guide the need for postoperative radiation therapyor chemotherapy in the setting of treatment de-intensification.Preliminary data demonstrates that HPV DNA levels in drain fluidmeasured through real-time PCR correlated with the number of positivelymph nodes in the neck (see FIG. 8A and FIG. 9 ) as well as withextracapsular extension of cancer in the lymph nodes (see FIG. 8B).Additionally, patients undergoing neck dissection but who are found tohave no pathologically-positive nodes show no detectable HPV in drainfluid. Further, patients undergoing bilateral neck dissectiondemonstrated different levels of HPV DNA within the samples obtainedfrom each of the two post-surgical drain lines that were correlated withthe nodal status of each side (see FIG. 10 ).

In various aspects, HPV DNA detected within drain samples may be used toguide radiation and chemotherapy dose after surgery as a potential meansof further de-intensifying treatment. In these various aspects,detection of HPV DNA in the drain fluid may be used as a biomarker,wherein patients with detected HPV DNA in the drain fluid may berecommended for treatment de-intensifying. Using drain fluid as a liquidbiopsy applies similar principles to those used to detect HPV cfDNA inplasma, but provides for a more proximal biomarker derived directly fromthe surgical bed. By way of non-limiting example, existing liquid biopsytests such as NavDx™ used for the detection of circulating tumor DNA(ctDNA) may be modified for use in detected cfHPVDNA in drainage fluidsamples.

In various aspects, the concept of using surgery drain fluid as aproximal marker to determine prognosis or adjuvant therapy for cancerscould be expanded to other cancer sites. In addition to head and neckcancer, HPV causes cervical, penile, and anal cancer. In variousaspects, HPV drainage fluids collected after surgical interventions ofother HPV-associated cancers may be analyzed to detect cfHPVDNA to aidin assessing patient prognosis and selected an appropriate treatmentplan. In another aspect, the methods described above may be used todetect other non-HPV biomarkers associated with other cancer typeswithin drainage fluid collected after surgical interventions. By way ofnon-limiting example, thyroglobulin, a known biomarker for thyroidcancer, may be detected in drainage fluid after surgical neck dissectionusing a detection method such as ELISA and used to assess patientprognosis and/or select a treatment plan for thyroid cancer.

In various aspects, a method for detecting minimal residual disease in asubject following a cancer surgery is disclosed. FIG. 1 is a flow chartillustrating the steps of the disclosed method in one aspect. The methodincludes obtaining a surgical drainage sample from the subject. Thesurgical drainage sample is obtained using any known method including,but not limited to, capturing a surgical drainage tube associated withthe cancer surgery. In some aspects, the sample may be obtained withinabout 24 hours of the completion of the surgery, providing thepractitioner with timely information regarding tumor-related geneticmaterial in the surgical drainage that may be used to select additionaltreatments.

In various aspects, the cancer surgery includes any surgery directed atremoving cancerous tissues or tumors from the subject including, but notlimited to, resectioning surgery, dissection surgery, excision surgery,and any combination thereof

Referring again to FIG. 1 , the method further includes isolating anamount of tumor-associated genetic material from the surgical drainagesample. In various aspects, the tumor-associated genetic materialincludes cell-free DNA, RNA, proteins, exosomes, and any combinationthereof. In other aspects, the tumor-associated genetic material isproduced or associated with a plurality of cancer cells. Non-limitingexamples of cancer cells include oropharyngeal cancer cells, lung cancercells, breast cancer cells, melanoma cells, colon cancer cells, thyroidcancer cells, prostate cancer cells, ovarian cancer cells, testicularcancer cells, penile cancer cells, cervical cancer cells, anal cancercells, brain cancer cells, liver cancer cells, pancreatic cancer cells,and testicular cancer cells.

Additional non-limiting examples of cancer cells include cells from avariety of cancer types including Acute Lymphoblastic Leukemia (ALL);Acute Myeloid Leukemia (AML); Adrenocortical Carcinoma; AIDS-RelatedCancers; Kaposi Sarcoma (Soft Tissue Sarcoma); AIDS-Related Lymphoma(Lymphoma); Primary CNS Lymphoma (Lymphoma); Anal Cancer; AppendixCancer; Gastrointestinal Carcinoid Tumors; Astrocytomas; AtypicalTeratoid/Rhabdoid Tumor, Childhood, Central Nervous System (BrainCancer); Basal Cell Carcinoma of the Skin; Bile Duct Cancer; BladderCancer; Bone Cancer (including Ewing Sarcoma and Osteosarcoma andMalignant Fibrous Histiocytoma); Brain Tumors; Breast Cancer; BronchialTumors; Burkitt Lymphoma; Carcinoid Tumor (Gastrointestinal); ChildhoodCarcinoid Tumors; Cardiac (Heart) Tumors; Central Nervous System cancer;Atypical Teratoid/Rhabdoid Tumor, Childhood (Brain Cancer); EmbryonalTumors, Childhood (Brain Cancer); Germ Cell Tumor, Childhood (BrainCancer); Primary CNS Lymphoma; Cervical Cancer; Cholangiocarcinoma; BileDuct Cancer Chordoma; Chronic Lymphocytic Leukemia (CLL); ChronicMyelogenous Leukemia (CML); Chronic Myeloproliferative Neoplasms;Colorectal Cancer; Craniopharyngioma (Brain Cancer); Cutaneous T-Cell;Ductal Carcinoma In Situ (DCIS); Embryonal Tumors, Central NervousSystem, Childhood (Brain Cancer); Endometrial Cancer (Uterine Cancer);Ependymoma, Childhood (Brain Cancer); Esophageal Cancer;Esthesioneuroblastoma; Ewing Sarcoma (Bone Cancer); Extracranial GermCell Tumor; Extragonadal Germ Cell Tumor; Eye Cancer; IntraocularMelanoma; Intraocular Melanoma; Retinoblastoma; Fallopian Tube Cancer;Fibrous Histiocytoma of Bone, Malignant, or Osteosarcoma; GallbladderCancer; Gastric (Stomach) Cancer; Gastrointestinal Carcinoid Tumor;Gastrointestinal Stromal Tumors (GIST) (Soft Tissue Sarcoma); Germ CellTumors; Central Nervous System Germ Cell Tumors (Brain Cancer);Childhood Extracranial Germ Cell Tumors; Extragonadal Germ Cell Tumors;Ovarian Germ Cell Tumors; Testicular Cancer; Gestational TrophoblasticDisease; Hairy Cell Leukemia; Head and Neck Cancer; Heart Tumors;Hepatocellular (Liver) Cancer; Histiocytosis, Langerhans Cell; HodgkinLymphoma; Hypopharyngeal Cancer; Intraocular Melanoma; Islet CellTumors; Pancreatic Neuroendocrine Tumors; Kaposi Sarcoma (Soft TissueSarcoma); Kidney (Renal Cell) Cancer; Langerhans Cell Histiocytosis;Laryngeal Cancer; Leukemia; Lip and Oral Cavity Cancer; Liver Cancer;Lung Cancer (Non-Small Cell and Small Cell); Lymphoma; Male BreastCancer; Malignant Fibrous Histiocytoma of Bone or Osteosarcoma;Melanoma; Melanoma, Intraocular (Eye); Merkel Cell Carcinoma (SkinCancer); Mesothelioma, Malignant; Metastatic Cancer; Metastatic SquamousNeck Cancer with Occult Primary; Midline Tract Carcinoma Involving NUTGene; Mouth Cancer; Multiple Endocrine Neoplasia Syndromes; MultipleMyeloma/Plasma Cell Neoplasms; Mycosis Fungoides (Lymphoma);Myelodysplastic Syndromes, Myelodysplastic/Myeloproliferative Neoplasms;Myelogenous Leukemia, Chronic (CML); Myeloid Leukemia, Acute (AML);Myeloproliferative Neoplasms; Nasal Cavity and Paranasal Sinus Cancer;Nasopharyngeal Cancer; Neuroblastoma; Non-Hodgkin Lymphoma; Non-SmallCell Lung Cancer; Oral Cancer, Lip or Oral Cavity Cancer; OropharyngealCancer; Osteosarcoma and Malignant Fibrous Histiocytoma of Bone; OvarianCancer Pancreatic Cancer; Pancreatic Neuroendocrine Tumors (Islet CellTumors); Papillomatosis; Paraganglioma; Paranasal Sinus and Nasal CavityCancer; Parathyroid Cancer; Penile Cancer; Pharyngeal Cancer;Pheochromocytoma; Pituitary Tumor; Plasma Cell Neoplasm/MultipleMyeloma; Pleuropulmonary Blastoma; Pregnancy and Breast Cancer; PrimaryCentral Nervous System (CNS) Lymphoma; Primary Peritoneal Cancer;Prostate Cancer; Rectal Cancer; Recurrent Cancer Renal Cell (Kidney)Cancer; Retinoblastoma; Rhabdomyosarcoma, Childhood (Soft TissueSarcoma); Salivary Gland Cancer; Sarcoma; Childhood Rhabdomyosarcoma(Soft Tissue Sarcoma); Childhood Vascular Tumors (Soft Tissue Sarcoma);Ewing Sarcoma (Bone Cancer); Kaposi Sarcoma (Soft Tissue Sarcoma);Osteosarcoma (Bone Cancer); Uterine Sarcoma; Sezary Syndrome (Lymphoma);Skin Cancer; Small Cell Lung Cancer; Small Intestine Cancer; Soft TissueSarcoma; Squamous Cell Carcinoma of the Skin; Squamous Neck Cancer withOccult Primary, Metastatic; Stomach (Gastric) Cancer; T-Cell Lymphoma,Cutaneous; Lymphoma; Mycosis Fungoides and Sezary Syndrome; TesticularCancer; Throat Cancer; Nasopharyngeal Cancer; Oropharyngeal Cancer;Hypopharyngeal Cancer; Thymoma and Thymic Carcinoma; Thyroid Cancer;Thyroid Tumors; Transitional Cell Cancer of the Renal Pelvis and Ureter(Kidney (Renal Cell) Cancer); Ureter and Renal Pelvis; Transitional CellCancer (Kidney (Renal Cell) Cancer; Urethral Cancer; Uterine Cancer,Endometrial; Uterine Sarcoma; Vaginal Cancer; Vascular Tumors (SoftTissue Sarcoma); Vulvar Cancer; or Wilms Tumor.

The tumor-associated genetic material may be isolated from the sampleusing any suitable method without limitation. Non-limiting examples ofsuitable isolation methods include filtering the sample, centrifugingthe sample, contacting the sample with a chromatography medium, and anycombination thereof

FIG. 2 is a schematic illustration of a method of isolating thetumor-associated genetic material from the sample in one aspect. Asillustrated in FIG.2, the surgical drainage is centrifuged and filtered.EDTA is added to the sample to inhibit nucleases in the sample. Thesample with added EDTA is further centrifuged, and the supernatant isremoved and retained. In some aspects, the supernatant may be used as isfor detection and quantification of cell-free DNA, RNA, and proteins. Inother aspects, illustrated in FIG. 2 , exosomes may be isolated from thesupernatant by contacting the supernatant with a chromatographic mediafollowed by elution. In various aspects, the isolation of thetumor-associated genetic material using the method illustrated in FIG. 2yields intact exosomes, as illustrated in FIG. 3A, FIGS. 5A-5J, and11A-11J. By contrast, other isolation methods such asultracentrification may damage the exosomes, as illustrated in FIG. 3A.

Referring again to FIG. 1 , the method further includes sequencing thetumor-associated genetic material in the sample anddetecting/quantifying tumor-associated mutations and/or variants. Anysuitable method may be used to sequence, detect, and quantify thetumor-associated mutations and/or variants including, but not limitedto, next generation DNA sequencing, next generation RNA sequencing, nextgeneration protein sequencing, PCR, Western blot, and any combinationthereof In some aspect, a targeted sequence assay panel may be used.

Referring again to FIG. 1 , the method may further include providing thequantities of detected tumor-associated mutations and/or variants to apractitioner. Based on these detected quantities, the practitioner maymake a determination of minimal residual disease in the patient, and/orselect a follow-up treatment. Non-limiting examples of suitablefollow-up treatments include radiotherapy, chemotherapy, follow-upsurgery, active surveillance with imaging, and any combination thereof

Definitions and methods described herein are provided to better definethe present disclosure and to guide those of ordinary skill in the artin the practice of the present disclosure. Unless otherwise noted, termsare to be understood according to conventional usage by those ofordinary skill in the relevant art.

In some embodiments, numbers expressing quantities of ingredients,properties such as molecular weight, reaction conditions, and so forth,used to describe and claim certain embodiments of the present disclosureare to be understood as being modified in some instances by the term“about.” In some embodiments, the term “about” is used to indicate thata value includes the standard deviation of the mean for the device ormethod being employed to determine the value. In some embodiments, thenumerical parameters set forth in the written description and attachedclaims are approximations that can vary depending upon the desiredproperties sought to be obtained by a particular embodiment. In someembodiments, the numerical parameters should be construed in light ofthe number of reported significant digits and by applying ordinaryrounding techniques. Notwithstanding that the numerical ranges andparameters setting forth the broad scope of some embodiments of thepresent disclosure are approximations, the numerical values set forth inthe specific examples are reported as precisely as practicable. Thenumerical values presented in some embodiments of the present disclosuremay contain certain errors necessarily resulting from the standarddeviation found in their respective testing measurements. The recitationof ranges of values herein is merely intended to serve as a shorthandmethod of referring individually to each separate value falling withinthe range. Unless otherwise indicated herein, each individual value isincorporated into the specification as if it were individually recitedherein. The recitation of discrete values is understood to includeranges between each value.

In some embodiments, the terms “a” and “an” and “the” and similarreferences used in the context of describing a particular embodiment(especially in the context of certain of the following claims) can beconstrued to cover both the singular and the plural, unless specificallynoted otherwise. In some embodiments, the term “or” as used herein,including the claims, is used to mean “and/or” unless explicitlyindicated to refer to alternatives only or the alternatives are mutuallyexclusive.

The terms “comprise,” “have” and “include” are open-ended linking verbs.Any forms or tenses of one or more of these verbs, such as “comprises,”“comprising,” “has,” “having,” “includes” and “including,” are alsoopen-ended. For example, any method that “comprises,” “has” or“includes” one or more steps is not limited to possessing only those oneor more steps and can also cover other unlisted steps. Similarly, anycomposition or device that “comprises,” “has” or “includes” one or morefeatures is not limited to possessing only those one or more featuresand can cover other unlisted features.

All methods described herein can be performed in any suitable orderunless otherwise indicated herein or otherwise clearly contradicted bycontext. The use of any and all examples, or exemplary language (e.g.“such as”) provided with respect to certain embodiments herein isintended merely to better illuminate the present disclosure and does notpose a limitation on the scope of the present disclosure otherwiseclaimed. No language in the specification should be construed asindicating any non-claimed element essential to the practice of thepresent disclosure.

Groupings of alternative elements or embodiments of the presentdisclosure disclosed herein are not to be construed as limitations. Eachgroup member can be referred to and claimed individually or in anycombination with other members of the group or other elements foundherein. One or more members of a group can be included in, or deletedfrom, a group for reasons of convenience or patentability. When any suchinclusion or deletion occurs, the specification is herein deemed tocontain the group as modified thus fulfilling the written description ofall Markush groups used in the appended claims.

Any publications, patents, patent applications, and other referencescited in this application are incorporated herein by reference in theirentirety for all purposes to the same extent as if each individualpublication, patent, patent application or other reference wasspecifically and individually indicated to be incorporated by referencein its entirety for all purposes. Citation of a reference herein shallnot be construed as an admission that such is prior art to the presentdisclosure.

Having described the present disclosure in detail, it will be apparentthat modifications, variations, and equivalent embodiments are possiblewithout departing the scope of the present disclosure defined in theappended claims. Furthermore, it should be appreciated that all examplesin the present disclosure are provided as non-limiting examples.

1. A method for detecting minimal residual disease in a subjectfollowing a cancer surgery, the method comprising: a. obtaining a samplefrom the subject, the sample comprising a surgical drainage; b.isolating an amount of tumor-associated genetic material from thesample; c. sequencing the amount of tumor-associated genetic material todetect and quantify at least one tumor-associated mutation or variant inthe amount of tumor-associated genetic material; d. providing the atleast one quantity of the at least one tumor-associated mutation orvariant to a practitioner, wherein the at least one tumor-associatedmutation or variant is indicative of minimal residual disease in thesubject.
 2. The method of claim 1, wherein the amount oftumor-associated genetic material comprises cell-free DNA, RNA,proteins, exosomes, and any combination thereof.
 3. The method of claim1, wherein isolating the amount of tumor-associated genetic materialfrom the sample further comprises filtering the sample, centrifuging thesample, contacting the sample with a chromatography medium, and anycombination thereof.
 4. The method of claim 1, further comprisingselecting an additional treatment based on the quantity of the at leastone tumor-associated mutation or variant.
 5. The method of claim 1,wherein the additional treatment is selected from radiotherapy,chemotherapy, follow-up surgery, active surveillance with imaging, andany combination thereof
 6. The method of claim 1, wherein thetumor-associated genetic material is produced by a plurality of cancercells.
 7. The method of claim 1, wherein the plurality of cancer cellsare selected from one of oropharyngeal cancer cells, lung cancer cells,breast cancer cells, melanoma cells, colon cancer cells, thyroid cancercells, prostate cancer cells, ovarian cancer cells, testicular cancercells, penile cancer cells, cervical cancer cells, anal cancer cells,brain cancer cells, liver cancer cells, pancreatic cancer cells, andtesticular cancer cells.
 8. The method of claim 1, wherein the cancersurgery is selected from a resectioning surgery, a dissection surgery,an excision surgery, and any combination thereof.
 9. The method of claim1, wherein obtaining the sample from the subject further comprisescapturing a surgical drainage from a drainage tube associated with thecancer surgery.
 10. The method of claim 1, wherein obtaining the samplefrom the subject further comprises capturing a surgical drainage fromthe drainage tube within about 24 hours of the cancer surgery.
 11. Themethod of claim 1, wherein the tumor-associated genetic materialcomprises cell-free HPV DNA (cfDNA) associated with oropharyngeal cancercells.
 12. The method of claim 1, wherein sequencing the amount oftumor-associated genetic material to detect and quantify at least onetumor-associated mutation or variant further comprises subjecting thesample to a sequencing method selected from next generation DNAsequencing, next generation RNA sequencing, next generation proteinsequencing, PCR, Western blot, and any combination thereof.
 13. Themethod of claim 1, wherein sequencing the amount of tumor-associatedgenetic material to detect and quantify at least one tumor-associatedmutation or variant in the amount of tumor-associated genetic materialfurther comprises detecting and quantifying at least one HPV strainassociated with HPV(+) oropharyngeal cancer comprising HPV 16 DNA, HPV18 DNA, HPV31 DNA, HPV33 fragment, HPV35 fragment, HPV45 DNA, HPV52 DNA,HPV58 DNA, and any combination thereof.
 14. A method for selecting apost-operative treatment for a cancer patient in need, the methodcomprising: a. obtaining a sample from the subject, the samplecomprising a surgical drainage; b. isolating an amount oftumor-associated genetic material from the sample; c. sequencing theamount of tumor-associated genetic material to detect and quantify atleast one tumor-associated mutation or variant in the amount oftumor-associated genetic material; d. providing the at least onequantity of the at least one tumor-associated mutation or variant to apractitioner, wherein the at least one tumor-associated mutation orvariant is indicative of minimal residual disease in the subject; and e.selecting an additional treatment based on the quantity of the at leastone tumor-associated mutation or variant.
 15. The method of claim 14,wherein the amount of tumor-associated genetic material comprisescell-free DNA, RNA, proteins, exosomes, and any combination thereof. 16.The method of claim 14, wherein isolating the amount of tumor-associatedgenetic material from the sample further comprises filtering the sample,centrifuging the sample, contacting the sample with a chromatographymedium, and any combination thereof.
 17. The method of claim 14, whereinthe additional treatment is selected from radiotherapy, chemotherapy,follow-up surgery, active surveillance with imaging, and any combinationthereof.
 18. The method of claim 14, wherein the tumor-associatedgenetic material is produced by a plurality of cancer cells.
 19. Themethod of claim 14, wherein the plurality of cancer cells are selectedfrom one of oropharyngeal cancer cells, lung cancer cells, breast cancercells, melanoma cells, colon cancer cells, thyroid cancer cells,prostate cancer cells, ovarian cancer cells, testicular cancer cells,penile cancer cells, cervical cancer cells, anal cancer cells, braincancer cells, liver cancer cells, pancreatic cancer cells, andtesticular cancer cells.
 20. The method of claim 14, wherein the cancersurgery is selected from a resectioning surgery, a dissection surgery,an excision surgery, and any combination thereof.
 21. The method ofclaim 14, wherein obtaining the sample from the subject furthercomprises capturing a surgical drainage from a drainage tube associatedwith the cancer surgery.
 22. The method of claim 14, wherein obtainingthe sample from the subject further comprises capturing a surgicaldrainage from the drainage tube within about 24 hours of the cancersurgery.
 23. The method of claim 14, wherein the tumor-associatedgenetic material comprises cell-free HPV DNA (cfDNA) associated withoropharyngeal cancer cells.
 24. The method of claim 14, whereinsequencing the amount of tumor-associated genetic material to detect andquantify at least one tumor-associated mutation or variant furthercomprises subjecting the sample to a sequencing method selected fromnext generation DNA sequencing, next generation RNA sequencing, nextgeneration protein sequencing PCR, Western blot, and any combinationthereof.
 25. The method of claim 14, wherein sequencing the amount oftumor-associated genetic material to detect and quantify at least onetumor-associated mutation or variant in the amount of tumor-associatedgenetic material further comprises detecting and quantifying at leastone HPV strain associated with HPV(+) oropharyngeal cancer comprisingHPV 16 DNA, HPV 18 DNA, HPV31 DNA, HPV33 fragment, HPV35 fragment, HPV45DNA, HPV52 DNA, HPV58 DNA, and any combination thereof.